Oxygen Consumption Rate‐Defined Phases Couple Metabolism to Matrix Dynamics in Chondrocyte‐Mesenchymal Stromal Cell Co‐Culture

Chondrocytes and mesenchymal stromal cells (MSC) metabolism shapes cartilage matrix quality, but cartilage engineering lacks a non-destructive, time-resolved readout that links oxygen uses to matrix assembly and mechanics under standard culture conditions. We continuously recorded oxygen-consumption rate (OCR) in nasal chondrocyte (NC), MSC, and NC-MSC co-culture pellets cultured for 27 days under normoxia, and integrated OCR trajectories with time-resolved gene expression, matrix histology, glycosaminoglycan (GAG)/DNA, and unconfined compression mechanics across defined NC:MSC ratios (monocultures; 3:1, 2:1, 1:1, 1:2, 1:3). OCR trajectories were reproducibly tri-phasic-(I) condensation/priming (Days 0-9), (II) differentiation/matrix synthesis (Days 9-21/24), and (III) maturation/remodeling (Days 24-27)-and strongly composition dependent. NC-rich mixtures exhibited an earlier hypoxic tone with transient HIF-1α, accelerated SOX9 followed by ACAN and COL2A1 induction, and marked GAG synergy peaking at 3:1. MSC-rich mixtures sustained late respiration with higher PGC-1α, elevated COL10A1 and MMP13, and achieved the highest equilibrium modulus at 1:3 despite lower GAG/DNA. An OCR downshift near Day 24 marked metabolic settling in most groups, whereas 1:3 pellets maintained or increased respiration, consistent with continued oxidative remodeling. Correlation analyses linked OCR features to hyaline anabolism in NC-rich pellets and to remodeling/hypertrophy in MSC-rich pellets, indicating ratio-specific coordination between respiratory and matrix-associated signatures. These findings support OCR monitoring as a sensitive, non-destructive process metric associated with chondrogenic stage transitions, provide guidance for selecting NC-rich ratios to maximize hyaline fidelity and GAG productivity, and MSC-rich ratios to increase stiffness while managing hypertrophic risk, and offer a generalizable framework for in-process bioenergetic control across tissue-engineering contexts.